1. Technical Field
The present invention relates to methods of separation and purification of trans-resveratrol and/or emodin from plant extracts with increased level of purity, as well as to nutraceutical and/or phytotherapic compositions containing the same. More precisely, the present invention relates to methods of separation and purification of the polyphenol trans-resveratrol and/or anthraquinone emodin of Polygonum cuspidatum and/or Rumex acetosa (Chinese plants) by selective extraction of solvents. The present invention also relates to phytomedicines prepared from roots and/or rhizomes of Rumex acetosa or from their fractions.
2. Related Art
The anthraquinone emodin (3-methyl-1,6,8-trihydroxyanthraquinone) is known for inhibiting the activity of several protein tyrosine kinases, such as p56lck, Her2-Neu and so forth. It is also known for inhibiting tumor cell growth, in various leukemic cell lines (U937, HL-60), and for playing a role in chemotherapy-resistance.
Trans-resveratrol (3,5,4′-tri hydroxystilbene) presents antioxidant, anti-inflammatory, antiviral, cardioprotective, neuroprotective, and chemoprotective activities. Besides protecting against infections and ischemia, it treats type 1 and 2 diabetes, reduces obesity and prevents aging. Resveratrol is a polyphenol found mainly in red grape skin and red wine. The more intense wine or grape color, the bigger the polyphenol content is. Studies seem to indicate that resveratrol can lower levels of low-density lipoproteins, also known as cholesterol LDL or “bad” cholesterol, and increase levels of high-density lipoproteins, cholesterol HDL, or “good” cholesterol. Especially when in an oxidized state, LDL can be retained in blood vessel walls resulting in the formation of atheroma plaques. These plaques cause atherosclerosis, which leads to blood vessel obstruction. Resveratrol stimulates HDL production by the liver, decreases LDL production and avoids circulating LDL oxidation, and therefore plays a role in lowering the risk of cardiovascular disorders, such as myocardial infarct.
The plant Polygonum cuspidatum (itadori, huzhang, Mexican-bamboo Japanese knotweed) comes from China and is known for having a good amount of resveratrol. In general, resveratrol may be separated and purified from the extracts of Polygonum cuspidatum by chromatography: a) in silica gel; b) in macroporous adsorption resin; c) in cellulose; d) in high-speed countercurrent; and e) in polyamide. These methods use sophisticated equipment and solvent mixtures that make solvent recovery difficult.
Emodin, as well, may be separated and purified from the extracts of Polygonum cuspidatum by chromatography: a) in silica gel; and b) in high-speed countercurrent. These methods use sophisticated equipment and solvent mixtures that make solvent recovery difficult.
The extracts of Polygonum cuspidatum sold nowadays in Brazil are imported, have a low concentration of trans-resveratrol (˜8%) and are contaminated by anthraquinone emodin (˜5%), which presents color and an undesirable side effect (laxative). In pharmaceutical formulations or food product compositions, where a concentration of 10-20% of trans-resveratrol is required, the presence of emodin is undesirable and inappropriate. Most methods of separation and purification use chromatographic systems that need expensive equipment and solvent mixture that make solvent recovery difficult. The present invention solves these problems by presenting simple and effective methods of separation and purification of polyphenol trans-resveratrol and/or emodin from different plant sources. Those skilled in the art and the manufacturers of pharmaceutical and/or food formulations use trans-resveratrol as an active and/or functional component in such formulations, referred to in the present invention as nutraceutical (pharmaceutical and/or food) compositions because of their properties and benefits. Some sources of trans-resveratrol are presented in Table 1.
TABLE 1Known sources of trans-resveratrol*SourceTrans-resveratrol conc.Grape (Vitis spp)0.16-3.54μg g−1Peanut (Arachis hypogaea)0.02-1.92μg g−1Pistachio (Pistacia vera)0.09-1.67μg g−1Common hop (Humulus lupulus)0.5-1μg g−1Blueberry (Vaccinium corymbosum)~32ng g−1Rhubarb (Rheum rhaponticum)3.9mg g−1Japanese knotweed0.524mg g−1(Polygonum cuspidatum)*Adapted from Nature Reviews Drug Discovery. 2006, 5: 493-506.
The present inventors, in the search for alternative and more available sources of trans-resveratrol and/or emodin, also developed a method for obtaining such actives from sorrel, whose use was not known for this purpose. Sorrel (Rumex acetosa), also called common or garden sorrel and spinach dock, is a perennial plant in the family Polygonaceae, with sagitate leaves, reddish flowers forming a panicle, and capsular fruits. Native of Europe and Asia, and exotic in Brazil, it is not taxonomically classified in Brazil (see http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=41241, or Global Biodiversity Information Facility, whose data are available at http://www.secretariat.gbif.net/portal/ecat_browser.jsp? termsASCCpted=true). It is cultivated because of its edible leaves, the red dye extracted from its roots, and some medicine uses presented in the present invention. Its leaves taste like vinegar due to the oxalic acid. In folk culture, it has anti-scurvy, laxative, and diuretic uses. In literature, the presence of trans-resveratrol in the roots of Rumex acetosa was not reported. Only hydroxyanthraquinones were identified and isolated from its roots (Agric. Biol. Chem. 1982, 46, 1913-1914). The only reports about trans-resveratrol in similar plants were described for Rumex bucephalophorus, a plant from Israel, in which trans-resveratrol was 0.165 mg/g (J. Nat. Prod. 2003, 66, 1270-1272; J. Agric. Food Chem. 2006, 54, 1243-1247).
Patent literature comprises some documents related to methods of separation and/or purification of emodin and/or trans-resveratrol. Although none of the documents anticipates or even indirectly suggests the inventive concept of the present invention, some of them are mentioned hereinbelow.
United States patent publication US 2006/0135797 relates to a method for obtaining diacerein with low aloe-emodin content. Initially, a weak base, preferably an organic one, is added to diacerein; then the aqueous solution of the salt formed is subjected to continuous or discontinuous extraction in a water-immiscible solvent (preferably ethyl and butyl acetate, toluene and xylene); and purified diacerein is precipitated by acidification. Although this method also uses emodin extraction with toluene, it is different from the present invention because it adds a weak base to diacerein before the extraction. On the other hand, in the present invention, the extract of Polygonum cuspidatum is just washed and dried to be extracted with toluene. Thus, although both methods have a common factor, they are different as a whole.
United States patent publication US 2004/0052879 describes an extraction method for products with pharmacological activity, especially those having antitumor activity, that come from spermatophyte plants. Such products are complex mixtures of compounds, such as cis-resveratrol and trans-resveratrol. Extraction is carried out in a neutral ambient with aliphatic alcohol, preferably methanol or ethanol. Solvent and material are stirred and the product is extracted in an oxygen-free and nitrogen saturated atmosphere. Final extract is then centrifuged, separated from the solid part, and concentrated under vacuum at low temperatures, with ethyl acetate or similar solvents.
United States patent publication US 2005/0008664 relates to formulations with emodin and/or its derivatives. More specifically, a lipid formulation contains emodin, providing emodin solubility in a pharmaceutical formulation.
Japan patent application JP 2005/281179A relates to a purification method for the extract of Reynoutria japonica Houtt using a liquid mixture of hydrophilic polar solvent, such as ethanol in water. Extract undergoes absorption with activated charcoal, and then desorption of activated charcoal by a polar solvent, in order to obtain resveratrol selectively with a high level of purity.
Document WO 01/03713A1 describes an extraction method for resveratrol and/or ε-viniferine in vines. This method comprises primary extraction and purification stages, preferably followed by secondary purification. Purified extract is distinguished by having a high content of resveratrol and/or ε-viniferine.
Among several technical advantages of the present invention are the separation and purification of emodin and/or trans-resveratrol with high levels of purity in a unique method and the preparation of phytomedicines from alternative and more available material.